Ras proteins regulate multiple cellular signaling pathways through interaction with an array of effectors including Raf kinases, phosphatidylinositol-3-kinase, Ral GEFs, and Nore1. The best characterized of these regulatory interactions occur on the cytoplasmic surface of the plasma membrane in response to signals from growth factor receptors. However, it is now apparent that Ras also signals from endomembrane compartments and distinct microdomains of the plasma membrane. This has heightened interest in defining the mechanisms of subcellular targeting of Ras and other small monomeric GTPases. We have focused on the subcellular targeting of Ras proteins as a model to understand how lipid modification contributes to protein trafficking and signaling. Ras proteins are posttranslationally modified by CaaX box prenylation (farnesylation), -aaX cleavage, methylation, and with the exception of K-Ras, palmitoylation. Posttranslational modification is required for subcellular targeting, but also plays a key role in the assembly of Ras-dependent signaling complexes in a spatially and temporally restrictive fashion. Palmitoylation is unique among lipid modifications in that it is readily reversible, controlled by the action of protein acyl transferases (DHHC PATs) and proposed acyl protein thioesterases (APTs). A model for Ras trafficking has been proposed that is based on cycles of acylation and deacylation coupled with membrane exchange (kinetic trap). The current proposal focuses on palmitoylation and depalmitoylation of yeast Ras proteins and determines how acylation/deacylation cycles control trafficking and signaling. Mutations in ras genes have been implicated in approximately 30% of all cancers, with the incidence Ras mutations appearing in some cancers being considerably higher. We have discovered that lipid modification of Ras proteins is required for Ras function and propose to study the role of lipid modification as a potential target of cancer chemotherapeutic drugs.